高毒力大丽轮枝菌特异片段SCF73突变株的构建及其致病力测定

Construction and pathogenicity tests of a mutated specific fragment SCF73 in Verticillium dahliae

摘要:

摘要:【目的】初步明确高毒菌株VDG1特异片段SCF73与大丽轮枝菌致病力的关系。【方法】通过比较基因组学分析和PCR鉴定，明确大丽轮枝菌高毒菌株VDG1相对于低毒菌株VDG2 的特异片段SCF73;构建SCF73片段敲除质粒，导入农杆菌AGL-1，应用农杆菌介导法转化大丽轮枝菌VDG1，抗性筛选和PCR扩增鉴定SCF73敲除转化子;利用果胶、纤维素和淀粉培养基模拟分析ΔSCF73降解细胞壁组分的能力，采用定量蘸根接种法鉴定其对感病棉种军棉1号的致病力。【结果】确定了大丽轮枝菌VDG1的特异片段SCF73，长度为27.1 kb，预测编码5个基因，推测2个基因具有水解酶功能;筛选获得了3个ΔSCF73突变株;突变株利用细胞壁组分的能力与野生型菌株VDG1 相比无显著差异;突变株对感病棉种军棉1号的致病力显著减弱。【结论】高毒力菌株VDG1 特异片段SCF73 在大丽轮枝菌致病过程中具有重要作用。

Abstract:

Abstract:［Objective］To identify preliminarily the specific fragment SCF73's function in Verticillium dahlia virulence．［Method］The specific fragment SCF73 exposed to be existed in the high-virulent V． dahliae strain VDG1 and not in the mild one VDG2． The SCF73 fragment was obtained from comparatively aligned genome sequences of the two strains and its existence was confirmed using PCR method． According to SCF73's DNA sequence，a homologous recombination plasmid was constructed to knock out the fragment． The Agrobacterium tumefaciens-mediated transformation technique was used to initiate the mutant ΔSCF73，followed by antibiotic resistance screening，and PCR verification． The mutant's ability to secrete carbohydrate hydrolase was analyzed using pectin，cellulose and starch media and its virulence to the susceptible cotton cultivar Gossypium hirsutum cv． Junmian1 was assessed．［Result］ SCF73 (27.1 kb) contains 5 genes，two of them have glycosyl hydrolase activity． Although the mutant ΔSCF73's carbohydrate hydrolase secretion was not significantly different from the control VDG1，virulence of the mutant to cotton plants decreased significantly accompanied with disease outburst delay．［Conclusion］The specific fragment SCF73 plays an important role in the virulence of V．dahlia towards its cotton host plants．